1. Field of the Invention
The present invention relates generally to the resolving of stereoisomers of quinidine and quinine. More particularly, the invention relates to the identification of the biological activity of the different stereoisomers of quinidine and quinine.
2. Description of the Related Art
Quinidine is the most prescribed anti-arrhythmic agent in the United States. However, the clinical utility of quinidine is limited by the adverse effect of diarrhea In addition, quinidine causes arrhythmias, especially the torsade de pointes variety that results from a long QT interval. The significant proarrhythmia (worsening of ventricular arrhythmias) associated with quinidine are possibly due to its combined effect on both the sodium depolarizing current and the potassium repolarizing current.
The first medicinal remedy for the treatment of cardiac arrhythmias is derived from the bark of the cinchona tree, indigenous to South America, where South American Indians long-used cinchona as medication. Europeans brought the remedy back from their New World explorations. Jean-Baptiste Senac, a French physician, is the first to describe the use of cinchona extract for cardiac irregularities (Willius, F A, et al., Proc. Staff Meet. Mayo Clin, 1942, 17, 294-296). Subsequently, a ship's captain with the medical condition auricular (atrial) fibrillation is seen by one of the leading European cardiologists, Professor Wenckebach, who had no treatment for the condition (Wenckebach, K F. Die unregelmassige Herztatigkeit und ihre klinische bedeutung. W. Engelmann, Leipzig, 1914). The ship's captain showed Wenckebach how the bark of the cinchona could control the cardiac irregularity. Following this, Wenckebach popularized the use of the cinchona extract for arrhythmia therapy. The principal active anti-arrhythmic ingredient of cinchona, quinidine, is identified in 1918 by the American chemist, Frey (Frey, W. et al., Wien. Klin. Wschr., 1918, 55, 849-853).
While quinidine is still actively prescribed in the United States, its use is severely limited by a number of problems. The drug prolongs the QT interval on the electrocardiogram, in a very heterogeneous way, creating a predisposition leading to the development of cardiac arrhythmias, especially Torsade de pointes, which is a rapid ventricular tachycardia. A number of studies have indicated that patients taking quinidine are at a higher risk for death than those not taking quinidine (e.g., Coplen S. E, et al., Efficacy and safety of quinidine therapy for maintenance of sinus rhythm after cardioversion; Circulation 1991; 84:527). Quinidine is also limited by severe GI disturbances with the most limiting side-effect being diarrhea. Recent research has shown that the drug affects both the cardiac sodium channel and the cardiac potassium channel, making quinidine a very complex “mixed” action agent (Krafte D. S., et al., Europ J Pharma 1994; 266, 245-254; Snyders D. J., et al., Molecular Pharma, 1991 41:322-330).
The anti-arrhythmic action of quinidine is thought to be due to its effect on the sodium channel. Quinidine is classified as a Vaughn Williams type Ia anti-arrhythmic. However, its prolongation of the QT interval due to APD (action potential duration) prolongation is not well understood. Subsequently, quinidine is found to show a significant effect in blocking the potassium repolarizing current, thus possessing type III Vaughn Williams (classification) effect. More recently the prolongation of APD has been found to be both an anti-arrhythmic action, as well as being the basis for the development of a rapid ventricular tachycardia with unique morphology called Torsade de Pointes ventricular tachycardia. Additionally, two potassium channels have been reported to be critically involved in human repolarization; one being IKr the rapid rectifier and the second being IKs, a slower ion channel in the human myocardium. While the anti-arrhythmic action of many agents on supraventricular arrhythmia and ventricular arrhythmias are moderated via inhibition of sodium channel (ex procainamide) the effect on the potassium channels (IKr and IKs) may be an important anti-arrhythmic action. However, strong IKr blocking action often leads to pro-arrhythmia of the Torsade de Pointes variety. Thus, a chiral isolate that has less IKr action, but retains sodium channel inhibition may offer considerably less pro-arrhythmia. Additionally, an agent causing less contractile augmentation of the GI smooth muscle may also be a significant advantage, since so many patients discontinue quinidine due to the diarrhea. Contractile augmentation may not be the only mechanism of quinidine induced diarrhea. A secretory action of quinidine may also be an important contributor to the agents diarrheal effects.
Quinidine has a duplicate drug in nature, quinine, the well known anti-malarial agent. Quinine is the chemical mirror image of quinidine, similar to the differences of the right from the left hand—identical but can't overlap, a structural characteristic in chemistry known as chirality. This results in quinidine and quinine being stereoisomers of each other. Quinine not only causes constipation, rather than diarrhea, but it affects cardiac ion channels a lot less than quinidine. These differences come about because of the mirror image relationships of the molecule at one chiral center. Thus, because two different stereoisomers exist that have different configurations, they cannot fit into receptors the same way. Since it is assumed that the anti-arrhythmic action, QT prolongation, and diarrhea occur as a result of the molecules acting at specific receptor sites such as ion channels, it would be anticipated that they have differed effects at different ion channels. In fact, stereoisomeric segregation of drug effects is a well established pharmacological strategy to determine if a drug acts at a receptor site. Thus, if one stereoisomer is active and the other is not, this is taken to mean that the molecule acts at a specific binding site. The fact that quinidine and quinine are stereoisomers, but have different properties implies that the diarrhea and anti-arrhythmic effects of these drugs occur through different binding sites and different mechanisms.
However, what is not recognized previously is that there are three additional chiral centers on the molecule that then create a total of sixteen isomers. Besides the optically active site at carbon 2 (C-2) position that segregates quinidine from quinine, there are three additional sties at the C-3, C-15 and C-20 carbon positions. Considering all the possibilities, there are eight possible isomers that have quinidine conformation at the C-2 position. Likewise, quinine also has eight possible isomers, together with quinidine making a total of sixteen possible stereoisomers.
It is expected that the different stereoisomers of quinidine and quinine have differential biological effects. For example, individual stereoisomers of quinidine or quinine could have different and specific effects on cardiac potassium channels or cardiac sodium channels. In addition, individual stereoisomers of quinidine or quinine could cause increased GI motility.
The expectation of differential effects of stereoisomers of quinidine or quinine is supported by the many known examples of different stereoisomers compounds having significantly different biological activity. For example, the different stereoisomers of beta-blockers (e.g. levalbuterol, and beta-amino alcohols), amphetamine (AP), methamphetamine (MAP), and penicillamine have different pharmacological activities and pharmacokinetic behaviors. The S-isomers of AP and MAP are each approximately five times more active on the central nervous system (CNS) than their respective R-isomer.
The commercial success of stereoisomers with specific biological activities is demonstrated by the antihistamine terfenadine, the psychoactive agent fluoxetine and the prokinetic gastrointestinal agent cisapride. Terfenadine is originally sold as a racemate mixture of R- and S-isomers under the name Seldane®. After discovering that racemic terfenadine is preferentially oxidized in rats to form a carboxylic acid metabolite enriched in the R-enantiomer, Hoechst Marion Roussel began marketing the R-isomer of terfenadine as Allegra® (fexofenadine). A single isomer preparation of fluoxetine (Prozac) is under development and a single isomer version of Zyrtec (cetirizine) may be available in the near future. A single isomer version of cisapride is marketed as norcisapride, which has a different receptor binding profile than the parent racemic drug.
Preliminary data regarding the pharmacodynamics of stereoisomers, such as that mentioned above, suggest that individual isomers can possess significant differences in receptor-binding profiles and follow different courses of absorption, distribution, metabolism and excretion. As such, the administration of single isomers may significantly reduce if not eliminate drug interactions mediated by the effect of stereoisomers on different biological receptors. Similar to other racemic compounds, it is expected that individual stereoisomers of quinidine and quinine are responsible for the diversity of effects displayed by such compounds (e.g., action on cardiac sodium and potassium channels as well as effects on GI motility). The ability to identify isomers of quinidine and quinine with differential effects on cardiac sodium channels, cardiac potassium channels, and GI motility would offer considerable potential clinical benefits. For example, specific stereoisomers of quinine or quinidine could be used as drugs for blocking only cardiac sodium channels or blocking only cardiac potassium channels while not causing diarrhea. Alternatively, a stereoisomers of quinine or quinidine not effecting the cardiac sodium or potassium channels, but increasing GI motility or mucosal secretion could be used as novel treatment for constipation or Gastroesophageal reflux disease (GERD). Therefore, the isolation of specific stereoisomers of quinidine and quinine could lead to a safer, less toxic and less pro-arrhythmic compounds than racemic quinidine.
The present invention provides for the isolation of quinidine and quinine stereoisomers. The present invention also provides assays for quantitative determination of optically active isomers of quinine and quinidine in biological fluids. The present invention also provides for assays for measuring the effects of stereoisomers of quinidine and quinine on cardiac potassium and sodium channels, as well as contractility and secretory assays for determining GI motility activity.